Oligonucleotides (ODN) containing the "CpG motif" account for the immune stimulatory activities of bacterial DNA. They are unusually effective mitogens for B cells because, in addition to driving the cells into and through cycle, they also inhibit apoptosis. Certain single base changes in ODN sequence can dramatically reduce activity. We have recently discovered that 3 of the ODN sequence variants actually inhibit cell cycle progress apoptosis protection and IL-6 secretion driven by stimulatory (ST-) ODN, whereas others are weak agonists or neutral in primary B cells. We have also shown that the order of potency of a series of stimulatory (ST-) ODN is the same for cycle entry, apoptosis protection, and IL-6 secretion implying a single control point for ODN signaling. Inhibitory (IN-) ODN block all the biologic effects as well as gene expression and transcription factor activation events (NFkB, AP-1, NF-IL-6) induced by ST-ODN, but not similar events induced by LPS or anti-CD40. Our application will determine the optimal base sequence for inhibition, and estimate the length of ODN sequence recognized by the CpG-recognizing molecule (RM). Spurred by the finding that an ODN with 2 motifs is more potent than ODN with 1, we will test whether bivalence or the sequence of "transplanted" motifs determine the activity of an ODN. Thus we may discover more potent ST- and IN-ODN than those now available. Based on evidence that IN-ODN act proximal to NFkB and AP-1, we will examine earlier events leading to NFkB or AP-1 to locate sites of action of IN-ODN. We have shown that 32P-labeled CpG-ODN bind a series of 4-5 proteins in B cell cytoplasmic extracts, and that the order of avidity of these proteins for ST-ODN matches their order of potency in biologic assays. We will test whether this is also true for IN-ODN, including whether the increased potency associated with having 2 motifs is reflected in avidity for CpG-binding proteins (BP). We will then obtain microsequence data on the most prominent proteins in an attempt to identify them. Toll-Like Receptor (TLR) 9 has recently been shown to be necessary for responses to ODN. Using a TLR9-transfected cell line we will test whether TLR9 or one of its associated proteins binds CpG-ODN directly and if it does, whether the avidity and EMSA mobility match one of the CpG-BPs. The importance of this project lies in the need to develop antidotes for excessive CpG stimulation potentially to be encountered in CpG vaccine trials, and to recognize and avoid IN-ODN motifs when constructing viral vectors for gene therapy and DNA vaccination. Possibly that IN-ODN motifs in mammalian DNA may keep autoimmune responses to endogenous ST-ODN motifs in check.